PROJECT SUMMARY/ABSTRACT Reactive oxygen species (ROS) are the byproduct of normal metabolism as well as the differential state and environment of the cell. They lead to oxidative stress and can be the causes of multiple pathologies, including cancer. Moreover, oxidative stress and the cell's ability to deal with it can play a major role in the treatment of these diseases. However, current methods to quantify oxidative stress in vivo are severely lacking and to date there is no routine method to non-invasively image redox or oxidative stress in humans. A new platform, hyperpolarized MRI, has the potential to change the way we interrogate metabolism in vivo. We and others have utilized the power of this approach, combined with endogenous substrates, to non-invasively image a metabolic substrate and its subsequent downstream products using conventional MRI. In our preliminary work, we have developed a novel approach to imaging oxidative stress using HP dehydroascorbate (HP DHA), the oxidized form of vitamin C. Using HP DHA, we are able to image the in vivo generation of HP vitamin C and utilize the cells' endogenous system to create a non-invasive redox measurement. Combining this with fast spectroscopic imaging approaches provides a means of readily imaging redox in mice. These exciting developments provide the basis for pursuing the objectives of this innovative proposal to utilize HP DHA MRI to further quantify oxidative stress in vivo. Using HP DHA, we will develop a parameter for conversion to Vitamin C in vivo that quantifies the amount of oxidative stress the cell is under using both acute generation of ROS in the normal brain and models of murine brain tumors treated with radiation. In parallel, we will utilize what we have recently learned about the physical chemistry of hyperpolarized probes to design a more robust and better performing HP DHA. Finally, we will conduct the optimization and toxicology studies necessary to translate HP DHA to humans, aiming to conduct the first-in-human studies of this approach. It is the overarching goal of this proposal to use this novel approach in metabolic imaging and lay the foundation for future metabolic imaging of oxidative stress in patients. This effort will also provide a means of non-invasively monitoring treatment response that aims to increase oxidative stress, which could be readily integrated into standard MRI.